Compounds that fluoresce have many uses and are known to be particularly suitable for biological applications where fluorescence is required for the detection of whole cells, cellular components, and cellular functions. For example, many diagnostic and analytical techniques require the samples to be fluorescently tagged so that they can be detected. This is achieved by using fluorescent dyes or probes which interact with a wide variety of materials such as cells, tissues, proteins, antibodies, enzymes, drugs, hormones, lipids, nucleotides, nucleic acids, carbohydrates, or natural or synthetic polymers to make fluorescent conjugates.
With synthetic fluorescent probes, ligands are frequently used to confer a specificity for a biochemical reaction that is to be observed and the fluorescent dye provides the means of detection or quantification of the interaction. These applications include, among others, the detection of proteins (for example in gels or aqueous solution), cell tracking, the detection of proteins via fluorescently labelled antibodies, the assessment of enzymatic activity, the staining of nucleic acids.
Long wavelength absorbance usually increases the utility of a fluorescent probe since it reduces the interference from cellular auto-fluorescence and reduces the cytotoxic effect of the fluorophore in combination with light. Although lasers are particularly useful as a concentrated light source for the excitation of fluorescence, at present the output of lasers is restricted to particular wavelengths of light. Compounds whose excitation spectra coincide with laser output are therefore of high utility. The argon laser is the most common light source for excitation of fluorescence, and has principal outputs, light at 488 nm and 514 nm. Fluorescent compounds that are excited by either of these wavelengths are therefore of particular utility. Alternatively, excitation of fluorescence can be achieved using solid state light sources such as Light emitting diodes. Fluorescent compounds excited by the light emitted from these alternative sources are also of particular utility.
Red fluorescent compounds are used extensively in many fields of biological study. Many of these, including Texas red, Tetramethyl rhodamine-isothiocyanate or red emitting BODIPY dyes require excitation at green wavelengths such as 542 nm. This limits their use in many applications, especially those where the argon-ion laser is used for excitation. Compounds such as ethidium bromide, can be excited with light from the argon-ion laser (520 nm band), but are not generally suitable for tagging of organic molecules other than nucleic acids. Other compounds such as phycoerythrin, can be excited using the argon-ion laser (488 nm) and does emit in the orange/red wavelengths. Phycoerythrin, however, has poor stability and a high molecular weight making it unsuitable for many applications such as cell tracking, labelling of nucleic acids or staining proteins.
For staining of proteins, there are a number of methods available. These methods can utilise non-fluorescent compounds, or fluorescent compounds. The most commonly used method utilises Coomassie blue which is non-fluorescent, can require the use of large amounts of organic solvents and is time consuming. Other fluorescence-based protein-detection methods are available which are potentially more sensitive than non-fluorescent methods. However, these methods are in general much more expensive than non-fluorescent methods which limits their widespread use. Therefore, compounds that combine useful spectral characteristics, and relatively high sensitivity will be of particular utility.
There are several methods for the quantification of protein in solution. These methods are based on a range of techniques, and include methods where dyes bind to soluble proteins. These dyes can be either non-fluorescent or fluorescent compounds. Fluorescent dye-based methods are often more sensitive than the non-fluorescent dyes, and allow for the determination of protein concentration over a wide range of concentrations. Compounds that combine useful spectral characteristics with an ability to bind proteins will be of particular utility.
In enzymatic studies, there is widespread use of fluorescent compounds for the detection of particular enzymatic activities. For example, fluorescein di-β-D-galactopyranoside (FDG) is a non-fluorescent compound that is sequentially hydrolysed by the enzyme .β.-galactosidase first to generate fluorescein monogalactoside and then to fluorescein which is highly fluorescent. The cleavage of the FDG compound can be monitored by the increase in fluorescence in the solution, and thus allows sensitive quantification of enzymatic activity. At present, only a limited number of fluorophores are suitable for this procedure. Therefore, novel fluorescent compounds that can be conjugated to a variety of substrates will be of utility.
For dual colour staining, there is a very limited choice of low molecular weight fluorophores. The predominant green fluorophore is fluorescein, which strongly absorbs light from the 488 nm band of the argon ion laser, and re-emits at 518 nm. At present there are few compatible red or orange fluorophores that are of low molecular weight and are excited by the 488 nm or 514 nm bands of the argon ion laser. Therefore, low molecular weight compounds that are excited by argon ion lasers and emit at wavelengths greater than 600 nm will be of utility, particularly if there is minimal spectral overlap with fluorescein.
The present inventors have isolated new compounds derived from a fungus that is capable of combining readily with a range of organic molecules to produce fluorescent complexes.